Cell-based therapy provides a minimally invasive approach by local injection at the site of defect via microsyringe needles and presents a promising approach for tissue repair and regenerative medicine. However, technological challenges associated with the localization, long term viability, host tissue-integration and functional remodeling (Tatard, et al., Curr. Drug Targets, 6(1):81-96 (2005); Tatard, et al., Biomaterials, 26(17):3727-37 (2005); Pittenger and Martin, Circ. Res., 95(1):9-20 (2004); Bonaros, et al., Panminerva Med., 46(1):13-23 (2004)) of the injected cells, and injectability and mechanical stability of the carriers (Crevensten, et al., Ann. Biomed. Eng., 32(3):430-4 (2004)) need to be resolved before clinical applications can be successfully achieved.
Microencapsulation entraps cells within the confinement of a semi-permeable membrane or a homologous solid. It has been used for many years to aid immunoisolation during allogenic or xenogenic cell transplantation (Uludag et al., Adv. Drug Deliv. Rev., 42(1-2):29-64 (2000); Orive, et al., Trends Biotechnol., 22(2):87-92 (2004)). Sodium alginate dominates the field while other materials such as agarose (Batorsky, et al., Biotechnol. Bioeng., 92(4):492-500 (2005)) and polyethylene glycol (PEG) (Nuttelman, et al., Matrix Biol., 24(3):208-18 (2005)) are also used. None of these materials, if unmodified, support cell attachment and growth (Grohn, et al., Biotechniques, 22(5):970-5 (1997); Zimmermann, et al., Biomaterials, 24(12):2083-96 (2003); Nuttelman, et al., Matrix Biol., 24(3):208-18 (2005)) thus requiring supplementation of natural extracellular matrix such as collagen for improvement (Grohn, et al., Biotechniques, 22(5):970-5 (1997); Batorsky, et al., Biotechnol. Bioeng., 92(4):492-500 (2005)). Furthermore, since these systems avoid direct contact of the delivered cells with the host tissue, they do not allow cell migration and penetration. This prevents their use in regenerative medicine and tissue engineering, which entails host-implant integration at cellular level. Natural extracellular matrices such as collagen, fibrin and hyaluronic acid are suitable materials supporting cell growth (Yannas, Natural Materials, Ratner, et al. editors, Biomaterials Sciences—An introduction to materials in medicine, California, Academic Press, pp. 84-93 (1996)). However, there is no microencapsulation system for these materials because of their poor mechanical and shape stability (Yannas, Natural Materials, Ratner, et al. editors, Biomaterials Sciences—An introduction to materials in medicine, California, Academic Press, pp. 84-93 (1996); Crevensten, et al., Ann. Biomed. Eng., 32(3):430-4 (2004); Zhang, et al., Appl. Biochem. Biotechnol., 134(1):61-76 (2006), which is incompatible with the existing microencapsulation techniques (Uludag, et al., Adv. Drug Deliv. Rev., 42(1-2):29-64 (2000)).
Existing encapsulation techniques include formation of emulsions with an oil phase and generation of cell-containing droplets in a stirred collection bath using a custom-made droplet generator, or injection of cells into preformed matrix microspheres or microcapsules using a microinjector (Grohn, et al., Biotechniques, 22(5):970-5 (1997); Batorsky, et al., Biotechnol. Bioeng., 92(4):492-500 (2005)). However, these methods encounter problems when the matrix materials are low in concentration, or with poor shape and mechanical stability such as collagen gel or hyaluoronic acid gel. First, the cell-matrix droplets or emulsions formed barely survive the shear stress generated upon stirring during emulsification or stirring in the liquid collection bath. Second, stirring is required immediately after addition of the cell-matrix phase to mix well with the oil phase during emulsification and to prevent the cell-matrix droplets from fusing together during droplet generation. This does not allow sufficient time for the formation of cell-matrix microspheres if the phase transition of the matrix takes a longer time and fragmentizes the microspheres, leading to low encapsulation efficiency.
Using living organisms with biosynthetic capability for large and industrial scale production of useful biomolecules such as therapeutic protein is commonly used in biotechnology. Although E. coli and yeasts have been used for this purpose, the resulting molecules may differ from the natural products because of the absence of co- and post-modifications mechanisms in these microorganisms. Mammalian cells are therefore particularly good sources for proteins and vaccines. Culturing cells in suspensions can attain high efficiency, reduce cost and favor mass production of therapeutic proteins. However, not all cells grow successfully in suspension, only cells such as hybridomas and tumor cells. Microcarrier technology has been developed for decades to enable large scale 3D culture by providing significantly increased surface area that is particularly advantageous for anchorage dependent eukaryotes. Microcarriers have been used for large scale cell culture as early as the 70's. The first generation microcarrier, CYTODEX®, dextran microspheres with a cationic surface, has been used to scale up cell cultures by dramatically increasing the total surface area for cell binding. The technology evolved in the 80's to include collagen-coated dextran beads, for better attachment and growth of cells and higher yield). There has been a trend in coating or mixing the solid microcarriers with natural extracellular matrix of cells such as collagen-coated alginate beads (Grohn, et al., Biotechniques, 22(5):970-5 (1997)), gelatin-coated poly-lactic-glycolic acid (PLGA) beads (Voigt, et al., Tissue Eng., 8(2):263-72 (2002)) and gelatin-chitin composite beads (Li, et al., Biotechnol. Lett., 26(11):879-83 (2004)), or crosslinking with natural peptide sequence governing cell adhesion and attachment such as RGD modified poly(ethylene glycol) (Nuttelman, et al., Matrix Biol., 24(3):208-18 (2005)) to improve cell attachment and growth. This has led to development of newer generations of microcarriers, CULTISPHER® G, which are either solid (Liu, et al., Cell Transplant., 13(7-8): 809-16 (2004)) or porous (Bancel and Hu, Biotechnol. Prog., 12(3):398-402 (1996)) gelatin beads, and CELLAGEN®, which are porous collagen beads (Overstreet, et al., In Vitro Cell Dev. Biol. Anim., 39(5-6):228-34 (2003)). However, these systems employ technologically demanding fabrication process for the bead preparations, making the commercial preparations costly. The bead preparation has to be separated from the cell attachment procedure since most of the bead fabrication systems employ harsh conditions such as high temperature, freeze-drying, organic solvent extraction and chemical crosslinking treatment that cells do not survive. Moreover, the cell attachment procedure is a rate-limiting step of the microcarrier culture system (Sun, et al., J. Biosci. Bioeng., 90(1):32-6 (2000)) requiring prolonged culture for cell attachment to the solid surfaces or cell penetration into the porous beads (Bancel and Hu, Biotechnol. Prog., 12(3):398-402 (1996)). As a result, simple bead preparation using natural extracellular matrix materials without harsh fabrication conditions and prolonged cell attachment procedurea will improve the efficiency and reduce the cost of the microcarrier culture system.
It is generally accepted that 3D culture provides a platform for cells to proliferate rapidly in an unrestricted manner (Geserick, et al., Biotechnol. Bioeng., 69(3):266-74 (2000)) for scaling up (Durrschmid, et al., Biotechnol. Bioeng., 83(6):681-6 (2003)). However, the productivity of actively and unrestrictedly proliferating cells is usually low because these cells may not synthesize proteins at a maximal rate outside their tissue-specific microenvironment and in actively proliferating cells, most of the metabolic energy is devoted to reproduction rather than synthetic activities (Sanchez-Bustamante, et al., Biotechnol. Bioeng., 93(1):169-180 (2005)). Controlled proliferation technologies such as starvation of cells for essential nutrient or addition of DNA synthesis inhibitors (Suzuki and Ollis, Biotechnol. Prog., 6(3):231-6 (1990)), isolation of specific cell lines such as temperature sensitive CHO cells, which produce more proteins upon temperature shift to 39° C. (Jenkins and Hovey, Biotechnol. Bioeng., 42(9):1029-36 (1993)) and genetic manipulation with growth cycle controlling genes such as over-expressing tumor suppressor genes p53 (Kastan, et al., Cancer Research, 51:6304-11 (1991)), p21 (Watanabe, Biotechnol. Bioeng., 77:1-7 (2002)) and p27 (Coats, et al., Science, 272:877-80 (1996)), are usually employed to enhance the protein productivity of cells (U.S. Pat. No. 6,274,341 to Bailey, et al.; Wurm, Nature Biotechnol., 2(11):1393-1398 (2004)). However, these proliferation controlling strategies lead to reduced cell viability (Mercille, et al., Cytotechnology, 15(1-3):117-28 (1994)) and increased apoptosis (Ko and Prives, Genes Dev., 10(9):1054-72 (1996)). Co-expressing the cell cycle controlling tumor suppressor genes with anti-apoptotic genes such as bcl-2 has been used to improve the cell viability problem (U.S. Pat. No. 6,274,341 to Bailey, et al.). However, this system requires complicated designs for the vector systems and complicated genetic manipulation that interferes the cell metabolism internally. Moreover, auto-regulated control for biphasic proliferation and production cycles has been achieved by using external repressable agent, tetracycline switch system (Mazur, et al., Biotechnol. Bioeng., 65:144-150 (1999)) to preserve the inducible growth-arresting production phase when the optimal cell density is reached so that a longer window for enhanced productivity for 7 days can be achieved. The advantages of this system include the use of external agents that do not interfere with the overall metabolism of cells but the problems are the downstream purification procedures eliminating this antibiotic, the genetic instability introduced by genetic manipulation as well as the instability of tetracycline in cultures. Recently, 3D multi-cellular micro-tissue cultures using a hanging-drop method with enhanced protein productivity in mammalian cells have been developed (Sanchez-Bustamante, et al., Biotechnol. Bioeng., 93(1):169-180 (2005)).
It is therefore an object of the invention to provide methods for making cell microcarriers that are relatively inexpensive, efficient and favorable to cell viability, controlled proliferation and production of biomolecules, and the resulting cell matrix microcarriers.
It is a further object of the invention to provide methods for use thereof in cell therapy and tissue engineering and manufacturing of biomolecules.